Project Summary/Abstract The proposed project will demonstrate the feasibility of generating a complete synapse-level brain map (connectome) by developing a serial-section electron microscopy pipeline that could scale to a whole mouse brain. This work will image 10 cubic millimeters, itself an unprecedentedly large dataset that may exceed tens of petabytes. Yet the mouse brain is 50 times larger. Reaching this ambitious goal will require advances in whole-brain staining, imaging, image-processing, analysis, and dissemination tools. We will scale and test these tools by producing a connectome of the hippocampal formation, a critical brain region for memory and spatial navigation. Specifically, we will define our volume of interest via microCT scanning of a whole brain. Then we will cut it into semithin serial sections and image them with multibeam scanning electron microscopy and ion beam milling. This technique images a thin layer of tissue and then removes it to reveal the next layer until each section is fully imaged, minimizing distortions caused by previous ultra-thin sectioning approaches. The imaging data will be processed by an improved version of our state-of-the-art pipeline. After quality monitoring and image compression, our automated system will assemble the full volume from imaged slices and then label tissue elements: neurons, glia, blood vessels, myelin, cell bodies, and synapses. This reconstruction will then be proofread and registered to the Allen Institute brain atlas, allowing us to relate our data to other types of data. Our analysis will identify cell types by region and layer, and reveal the detailed connectivity of hippocampal formation circuits. Using custom software, we will integrate these structural results on cell types with other approaches based on light microscopy and single-cell gene expression, allowing us to relate our results to the extensive literature on hippocampal formation structure and function. To promote diverse perspectives, we will involve undergraduates from underrepresented backgrounds in the proofreading and scientific discovery phases of our work, offering them mentoring as well as research experience. We will turn these data into a lasting resource for the scientific community and the public by scaling up free access via online sharing tools to allow any interested party to render, proofread, or otherwise analyze the cells and circuits in this volume. To illustrate how this resource can be combined with other discoveries, we will define cell types based on their morphology and connectivity, characterize the relationship between these assignments and transcriptomic-based classifications, and integrate this information with previous work. Finally, we will define local and long-range microcircuit motifs in our data and use it to identify circuit principles and mechanisms of memory and spatial cognition, by testing and improving models of the hippocampal formation. Throughout the project, we will monitor key performance parameters, such as imaging throughput of a single microscope, to evaluate the feasibility and cost of scaling up to a whole mouse brain connectome.

Project Narrative The brain’s neural circuitry (connectome), which underlies behavior and presumably goes awry in disorders of the nervous system, is largely unexplored in mammals. The proposed project aims to produce a large-scale connectome that includes all the main areas of the mouse hippocampal formation. This region is of clinical interest because it is an essential part of the circuit underlying spatial navigation and memory and the earliest impairments and degeneration related to Alzheimer’s disease. the site of